The fundamental objective of the proposed research is to establish a basic level of knowledge concerning the detailed molecular structure of the enzyme dihydrofolate reductase. It is expected that this enzyme's structural variability from species to species will provide a basis for rational development of species specific chemotherapeutic drugs. We expect to solve the X-ray crystal structure of an L. casei dihydrofolate reductase-methotrexate-NADPH ternary complex using a recently obtained electron density map. Mechanism arguments and a rationalization of inhibitor binding will be developed based on: (1) structural comparison between E. coli and L. casei enzymes, (2) small molecule binding studies using the powerful difference Fourier technique, and (3) a postulated structure for a mammalian dihydrofolate reductase derived from primary sequence comparisons with the two bacterial enzymes. In addition, preliminary crystallization experiments with S-180 dihydrofolate reductase from mouse sarcoma suggest that it may also be possible experimentally to determine this structure by X-ray crystallographic techniques.